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Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays

The large unmet demand for new heart failure therapeutics is widely acknowledged. Over the last decades the contractile myofilaments themselves have emerged as an attractive target for the development of new therapeutics for both systolic and diastolic heart failure. However, the clinical use of myo...

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Autores principales: Parijat, Priyanka, Ponnam, Saraswathi, Attili, Seetharamaiah, Campbell, Kenneth S., El-Mezgueldi, Mohammed, Pfuhl, Mark, Kampourakis, Thomas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10063609/
https://www.ncbi.nlm.nih.gov/pubmed/36997544
http://dx.doi.org/10.1038/s41598-023-32476-w
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author Parijat, Priyanka
Ponnam, Saraswathi
Attili, Seetharamaiah
Campbell, Kenneth S.
El-Mezgueldi, Mohammed
Pfuhl, Mark
Kampourakis, Thomas
author_facet Parijat, Priyanka
Ponnam, Saraswathi
Attili, Seetharamaiah
Campbell, Kenneth S.
El-Mezgueldi, Mohammed
Pfuhl, Mark
Kampourakis, Thomas
author_sort Parijat, Priyanka
collection PubMed
description The large unmet demand for new heart failure therapeutics is widely acknowledged. Over the last decades the contractile myofilaments themselves have emerged as an attractive target for the development of new therapeutics for both systolic and diastolic heart failure. However, the clinical use of myofilament-directed drugs has been limited, and further progress has been hampered by incomplete understanding of myofilament function on the molecular level and screening technologies for small molecules that accurately reproduce this function in vitro. In this study we have designed, validated and characterized new high throughput screening platforms for small molecule effectors targeting the interactions between the troponin C and troponin I subunits of the cardiac troponin complex. Fluorescence polarization-based assays were used to screen commercially available compound libraries, and hits were validated using secondary screens and orthogonal assays. Hit compound-troponin interactions were characterized using isothermal titration calorimetry and NMR spectroscopy. We identified NS5806 as novel calcium sensitizer that stabilizes active troponin. In good agreement, NS5806 greatly increased the calcium sensitivity and maximal isometric force of demembranated human donor myocardium. Our results suggest that sarcomeric protein-directed screening platforms are suitable for the development of compounds that modulate cardiac myofilament function.
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spelling pubmed-100636092023-04-01 Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays Parijat, Priyanka Ponnam, Saraswathi Attili, Seetharamaiah Campbell, Kenneth S. El-Mezgueldi, Mohammed Pfuhl, Mark Kampourakis, Thomas Sci Rep Article The large unmet demand for new heart failure therapeutics is widely acknowledged. Over the last decades the contractile myofilaments themselves have emerged as an attractive target for the development of new therapeutics for both systolic and diastolic heart failure. However, the clinical use of myofilament-directed drugs has been limited, and further progress has been hampered by incomplete understanding of myofilament function on the molecular level and screening technologies for small molecules that accurately reproduce this function in vitro. In this study we have designed, validated and characterized new high throughput screening platforms for small molecule effectors targeting the interactions between the troponin C and troponin I subunits of the cardiac troponin complex. Fluorescence polarization-based assays were used to screen commercially available compound libraries, and hits were validated using secondary screens and orthogonal assays. Hit compound-troponin interactions were characterized using isothermal titration calorimetry and NMR spectroscopy. We identified NS5806 as novel calcium sensitizer that stabilizes active troponin. In good agreement, NS5806 greatly increased the calcium sensitivity and maximal isometric force of demembranated human donor myocardium. Our results suggest that sarcomeric protein-directed screening platforms are suitable for the development of compounds that modulate cardiac myofilament function. Nature Publishing Group UK 2023-03-30 /pmc/articles/PMC10063609/ /pubmed/36997544 http://dx.doi.org/10.1038/s41598-023-32476-w Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Parijat, Priyanka
Ponnam, Saraswathi
Attili, Seetharamaiah
Campbell, Kenneth S.
El-Mezgueldi, Mohammed
Pfuhl, Mark
Kampourakis, Thomas
Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays
title Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays
title_full Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays
title_fullStr Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays
title_full_unstemmed Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays
title_short Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays
title_sort discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10063609/
https://www.ncbi.nlm.nih.gov/pubmed/36997544
http://dx.doi.org/10.1038/s41598-023-32476-w
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